The present invention relates to a novel process for the isolation of the pharmaceutically acceptable alkali metal salt of clavulanic acid from a fermentation broth containing impure clavulanic acid comprising the steps of filtration of the fermented broth, extraction of clavulanic acid to a water immiscible or partly water immiscible solvent at a pH from 1.2 to 2, precipitation of the alkali metal salt A of clavulanic acid by addition of a solution of an alkali metal alkylalkanoate.
Clavulanic acid and its alkali metal salts and esters are used in pharmaceutical preparation to prevent the deactivation of xcex2-lactam antibiotics. Commercial preparations of clavulanic acid contain potassium clavulanate in combination with amoxycillin trihydrate. Clavulanic acid is an unstable hygroscopic oil. Potassium clavulanate is more stable than the free acid or other salts, and is therefore most frequently, used for commercial preparations.
Clavulanic acid and its derivatives are inhibitors of the xcex2-lactamases. The resistance of xcex2-lactam antibiotics is associated with inactivation of xcex2-lactam structure due to the opening of xcex2-lactam ring by xcex2-lactamase produced by bacteria. Thus the inactivating enzymes are commonly called as xcex2-lactamase, they are divided into penicillinase and cephalosporinase.
Furthermore clavulanic acid itself is believed to have an antibacterial activity.
Clavulanic acid is produced from various strains of microorganism by a fermentation process. For this process for example strains belonging to the genus Streptomyces such as S. clavuligerus NRRL 3585 (U.S. Pat. No. 4,110,165), S. jumonjinensis NRRL 5741 (British Patent 1,563,103), S. katsurahamanus IFO 13716 (Japanese Patent 83,009,579), and Streptomyces sp. P6621 FERM 2804 (Japanese Patent 55,162,993) are used. For the preparation of clavulanic acid by a fermentation process the microorganism Streptomyces clavuligerus is preferred.
In the state of art different processes for the preparation and purification of the clavulanic acid containing fermentation broth are described.
GB 1 508 977 discloses preparation of clavulanic acid and its salts by filtration of the fermentation broth by passage through an anionic exchange resin.
GB 1 543 563 discloses a fermentation process wherein the pH value of the medium is maintained in the range of 6.3 to 6.7. A pharmaceutically acceptable salt such as potassium clavulanate is prepared by ion exchange process from lithium clavulanate. However lithium clavulanate is not a pharmaceutically acceptable salt. Therefore it is necessary to add an ion exchange process as a further step for preparing a pharmaceutically acceptable form of the compound. Furthermore, the remaining salt lithium chloride is soluble in organic solvents and therefore it is difficult to separate the lithium chloride in the aqueous phase during the extraction process.
Further documents of the state of the art like EP-0 647 229 describe the use of amine salts of clavulanic acid as intermediate compounds for the preparation and purification of clavulanic acid and its alkali metal salts. EP-0 647 229 for example describes a process for the preparation of a purified clavulanic acid or a salt or ester thereof by preparing a diamine salt of clavulanic acid and converting this intermediate compound into clavulanic acid or a pharmaceutically acceptable salt or ester. The conversion is made by adding for example potassium 2-ethylhexanoate and precipitating potassium clavulanate. Many of such amines are either unsuitable for the production of a salt of clavulanic acid or they give rise to amine salts of clavulanic acid which are either hygroscopic or toxic or both and, therefore, are unsuitable for use as intermediates for the preparation of a pharmaceutically acceptable compound.
Other purification processes of the state of the arts are performed without any amine compounds. For example WO 95/34194 A2 describes a process for manufacturing an alkali metal salt of clavulanic acid wherein impure clavulanic acid in aqueous solution is extracted by a solvent mixture of ketone and alkyl acetate under acidic condition. The solution is than treated in a conventional manner and the solution of an alkali metal salt of alkanoic acid dissolved in ketone or alkanol solvent is added to obtain pure alkali metal salt of clavulanic acid. Thus the process according to this state of the art omits the step of formation of amine salts. This process has the advantage that the use of mostly toxic amines is no longer necessary. In a preferred embodiment as alkali metal salt of alkanoic acid sodium or potassium salts are used, especially potassium 2-ethylhexanoate.
A similar process is also described in WO 96/28452 A1. This process comprises the steps of removing solids from a clavulanic acid containing fermentation broth by microfiltration, acidifying the filtrate to a pH between 1 and 3, extracting the acidified filtrate with a water immiscible solvent and separating the clavulanic acid containing extract. This extract is mixed with a metal donor and at least one additional solvent. From the solution the metal clavulanate salt is separated.
As metal donor compounds organic salts, carbonates, bicarbonates or hydroxides of potassium, sodium, lithium or magnesium can be used. The use of carboxylic acid salt is preferred. Further preferred metal donors include potassium 2-ethylhexanoate, potassium acetate, lithium 2-ethylhexanoate and lithium acetate.
EP-0 182 522 B1 also describes a process for the preparation of clavulanic acid and its salts and esters. In this process the fermentation broth is worked up as follows. The solids are removed by filtration or centrifugation. The broth is acidified to a pH of 1 to 3 and clavulanic acid is extracted by adding a water immiscible solvent with two phases being separated for example by centrifugation. This gives the clavulanic acid in the water immiscible phase. The solution is purified by mixing it with the dissolved lithium 2-ethylhexanoate solution isolating lithium clavulanate and optionally converting the lithium salt to other salts or an ester. The conversion of the lithium salt to other salts is carried out by ion exchange procedures using ion exchange resins in the form of the desired cation preferably sodium or potassium.
The processes of the state of the art referred above have the disadvantage, that the alkali metal salts prepared by direct precipitation are not pure enough for a pharmaceutical use. Therefore, further purification steps like recrystallization, purification over a column etc. are necessary.
The object of the invention therefore is to prepare clavulanic acid and its pharmaceutically acceptable alkali metal salts such as potassium clavulanate in a new and simple manner wherein the desired substance is obtained in a very high yield without any additional purification steps and of high purity avoiding the use of toxic amines or lithium compounds.
This technical problem is solved by a process which is characterized by the following steps:
before the filtration of the fermentation broth, the fermentation broth containing clavulanic acid is diluted with water, a flocculating agent is added and the pH is adjusted to pH 3 to 5,
for further purification the alkali metal salt A of clavulanic acid is converted to clavulanic acid by addition of an inorganic acid and is extracted into a water immiscible or partly water immiscible solvent,
and to the solution of clavulanic acid a solution of a different alkali metal alkylalkanoate B is added and the alkali metal salt B of clavulanic acid is precipitated.
Suitable salts according to the present invention are pharmaceutically acceptable alkali metal salts and alkaline earth metal salts like sodium, potassium, calcium and magnesium salts.
Of these compounds potassium clavulanate is the most stable compound which is normally used for pharmaceutical preparations. The clavulanic acid itself is an unstable hygroscopic oil which is not used for the preparation of pharmaceutical compounds.
In a preferred embodiment the alkylalkanoate is an alkylhexanoate, especially 2-ethylhexanoate. The alkali metal A is sodium and the alkali metal B is potassium.
In a further preferred embodiment the filtered broth containing clavulanic acid is purified after the filtration step by adsorption on an anion exchange resin containing column and eluted with an aqueous solution of an alkali metal salt. As anionic exchange resin for example DIAION(copyright) SA-11A is used. After the elution step clavulanic acid is extracted into a water immiscible solvent or a partly water immiscible solvent and sodium clavulanate is precipitated by the addition of sodium 2-ethylhexanoate solution in an appropriate solvent after dehydration with anhydrous sodium or magnesium sulphates and purification with activated carbon.
As an alternative process step it is also possible to carry out the direct extraction of the clavulanic acid contained in the filtered broth to a water immiscible or partly water immiscible solvent at an adequate pH between 1.2-2.0 without using a column. Preferably 3 to 4 volumes of solvent in relation to the filtered broth are used in this step. The clavulanic acid is extracted from the above solvent to an aqueous solution using an organic base, preferably triethylamine or diethylamine. In this operation, a concentration of about 10 to 15 times is obtained. The exhaust solvent can be re-used in the process without a purification step. The clavulanic acid is back extracted to a water immiscible or partly water immiscible solvent. From the solvent sodium clavulanate is precipitated by addition of the sodium 2-ethylhexanoate solution in an appropriate solvent after dehydration.
Clavulanic acid is a hygroscopic oil and is not very stable in aqueous solution. Therefore, this solution of clavulanic acid in a water immiscible or partly water immiscible solvent is dehydrated in a preferred manner by addition of anhydrous sodium or magnesium sulphate and further purified by addition of activated carbon. By the use of the activated carbon the coloured impurities are removed from the solution.
In a preferred embodiment as solvent ethyl acetate, butyl acetate, methyl isobutyl ketone or mixtures thereof are used. Furthermore, before the filtration step a flocculating agent can be used. As flocculating agents quaternary ammonium salts are preferred.
A further object of the present invention is the use of sodium clavulanate as intermediate compound for the preparation of the pharmaceutically acceptable potassium clavulanate.
Filtration of clavulanic acid fermented broth is normally difficult and the use of flocculating agents has been suggested to improve filterability of the whole broth as for example described in EP-A 0 387 178. The present invention describes a new simple and cheap method to improve not only the filterability of the whole broth but also a means of increasing the yield of filtration and a facilitation of the subsequent down stream process.
Surprisingly it has been found possible to improve the recovery of clavulanic acid from an aqueous fermented broth by pre-diluting it with water. This pre-dilution of the fermented broth when combined with the use of flocculating agents and pH-adjustment is even more efficient, both at the level of filterability and yield of filtration and subsequent down stream operations. The pre-dilution of the broth with water decreases the viscosity of the same making the filtration easier. The yield of filtration is also improved since a poorer filtrate remains in the filter cake. The use of a flocculating agent and the gentle acidification to a pH value of 3 to 5 leads to precipitation of proteins which will be retained in the filter cake improving the filtration rate. In this way, a purer filtrate is obtained implying that the further recovery operations for the clavulanic acid are much easier. According to the present invention high quality potassium clavulanate can be produced with good yields from the fermented broth of clavulanic acid producing microorganisms through the steps which are described in the characterizing part of claim 1.
After filtration of the fermented broth the solution can be directly extracted to an organic solvent at an adequate pH is about 1.2 to 2.0, preferably using 3 to 4 volumes of solvent in relation to the filtered broth. It is also possible to adsorb the solution before that step onto an anionic exchange resin and to elute clavulanic acid with an aqueous solution of an alkaline metal salt. The extraction step leads to an organic phase which contains the clavulanic acid from the fermentation process. The organic phase is dehydrated with anhydrous sodium or magnesium sulphate and purified with activated carbon. Thereafter sodium 2-ethylhexanoate in an organic solution is added and after a period of crystallization crystals of sodium clavulanate can be collected by filtration.
The conversion from sodium clavulanate to potassium clavulanate is carried out by extraction of clavulanic acid to an adequate solvent and crystallization of potassium clavulanate after dilution of the acid with an adequate solvent and addition of potassium 2-ethylhexanoate or potassium acetate solution in an appropriate solvent. For this process step sodium clavulanate is suspended in a mixture of methyl isobutyl ketone or ethyl acetate or butyl acetate with water. To this solution an inorganic acid is added. By this addition the salt of the clavulanic acid is converted into clavulanic acid. The acid is extracted into the organic phase with stirring.
The mixture is then diluted with isopropanol and a solution of potassium 2-ethylhexanoate in isopropanol is added to reach a pH between 6 and 7. After a crystallization period of 2 hours at low temperature potassium clavulanate crystals can be collected by filtration.
If necessary potassium clavulanate can be recrystallized.
In a further preferred embodiment it is possible to extract the clavulanic acid from the water immiscible or partly water immiscible solvent to an aqueous solution using an organic phase preferably triethylamine or diethylamine. By this operation concentration of about 10 to 15 times can be obtained. The exhaust solvent can be reused in the process without any purification step.
A further alternative way is a back extraction of clavulanic acid to the water immiscible or partly immiscible solvent.
By the process of the present invention clavulanic acid salts of potassium can be prepared in high purity and good yields. The process is simple and allows to work without the use of any toxic amines. Furthermore the purity of the potassium clavulanic acid prepared by the present invention is higher then the purity of this compound made from processes of the state of the art which directly use the precipitation by reaction with potassium 2-ethylhexanoate.
The following examples are intended to illustrate the present not to limit it.
A. Experiments of Filtration of Fermented Broth of Clavulanic Acid